CN117890597A - Anti-drug antibody detection system and method for IgE targeted drug - Google Patents

Abstract

The invention belongs to the technical field of biological detection, and discloses an anti-drug antibody detection system and method of an IgE targeted drug, wherein the detection system comprises a sample processing system and a detection system, and the sample processing system comprises microbeads coupled with anti-human IgE antibodies and dissociation neutralizing reagents; the detection system comprises an ELISA plate coated with the omalizumab, biotin-labeled omalizumab, ruthenium-labeled omalizumab and an MSD plate coated with streptavidin. The system and the method are used for detecting the anti-drug antibody of the IgE targeted drug, and the detection sensitivity can reach 0.69ng/mL.

Description

Anti-drug antibody detection system and method for IgE targeted drug

Technical Field

The invention belongs to the technical field of biological detection, and relates to a detection system and a detection method for an anti-drug antibody of an IgE targeted drug.

Background

Allergic disease is a hypersensitivity disease, and common are atopic dermatitis, allergic rhinitis and allergic asthma. When an individual is exposed to an allergen, activated B cells differentiate into plasma cells, synthesizing and secreting IgE. The heavy chain constant region (Fc fragment) of IgE binds mainly to the high affinity receptor (fceri) of IgE receptors on the surface of effector cells, mediating the allergic inflammatory cascade leading to inflammation in individuals and possibly life threatening when severe. It follows that IgE (immunoglobulin E) plays a critical role in hypersensitivity reactions, and is a key point in drug therapy and a preferred target for drug development.

Taking amazuki developed by the cooperation of North China and Roche as an example, the amazuki which is the first IgE targeted therapeutic drug for treating allergic asthma worldwide, the accumulated sales amount exceeds 300 hundred million dollars since the market, and the market performance is unusual. The success of the IgE target has proved the effectiveness of the IgE target in allergic diseases, reflects the huge market potential of IgE targeted drugs, and greatly enhances the confidence of developing the IgE targeted drugs for various manufacturers. Currently, many IgE-targeted drugs of manufacturers enter clinical test stages, such as nowa (Ligelizumab, clinical third-phase), zhangjiang organisms (CMAB-007: aomiaishu, clinical third-phase), rouse (quillizumab, clinical second-phase), heaven organisms (humanized human IgE monoclonal antibody injection, clinical first-phase), and the like, and more IgE-targeted drugs are developed and optimized in the layout of pharmaceutical manufacturers in the future, so that research on IgE-targeted drugs has become a research hotspot for allergic diseases.

The IgE targeted drug is used as a macromolecular drug, has immunogenicity, can induce individual immune response, and generates an Anti-drug Antibody (ADA), on one hand, the biological activity of the drug can be possibly neutralized, so that the drug effect is obviously reduced; on the other hand, cross-reactions with endogenous proteins may occur, leading to new allergic reactions, cytokine release, etc., and in severe cases, life may be endangered. Therefore, detection and characterization of ADA is essential in the whole drug development life cycle, so that establishment of an ADA detection method with sufficient sensitivity and high specificity is extremely important for preclinical and clinical analysis and evaluation of drugs.

IgE targeted drug targets IgE are secretory immunoglobulins, the highest content in healthy individuals can reach 873.4ng/mL, and the concentration in allergic patients can be obviously improved by more than 10 times, and the change is obvious. In the detection process of ADA, the IgE targeting drug is generally taken as a capture reagent, and the specific binding capacity of the IgE targeting drug and IgE is extremely strong, so IgE can compete with ADA for binding the capture reagent, and even the concentration change of the IgE can affect the detection result to different degrees. Meanwhile, the IgE levels of different individuals are different, and the half-life period of IgE is short and the stability is poor, so that the sensitivity, accuracy, application range and stability of the detection method can be further reduced.

For interference removal in the ADA detection process, interference removal of free drugs is studied in many cases. In chinese patent CN109358192a, a device and method for removing free drug from a sample for detection of an anti-drug antibody is disclosed, which is characterized in that the device couples a substance specifically binding to the drug to a solid carrier, and the substance does not react with other components than the free drug. The inventor well solves the problem of drug interference in ADA analysis by using the drug adsorption device, but according to the data calculation of the application example, the adsorption device has the adsorption clearance of about 32% at 10 minutes and about 33.3% at 20 minutes when carrying out adsorption test on a sample with the drug concentration of 4.41mg/mL, so that the adsorption clearance is about 30% and does not change significantly with time. Although the main difference between the free drug, capture reagent and detection reagent is the labeling molecule or treatment: the free medicine is in a free state without treatment; the capture reagent may be chemically coupled to the microbeads or physically adsorbed to the well plate; the detection reagent is marked with biotin, fluorescein or ruthenium, but the binding capacity of the three substances and the target substance is not substantially different. When the concentration difference of the three is less than 10 times, the reaction still has competition, but the detection result is in direct proportion to the ADA concentration in the sample, so that the qualitative or semi-quantitative detection result can be obtained. The IgE targeted drug is a product developed by manual screening, has strong specific binding capacity with IgE, and can be that a capture reagent is firstly bound with IgE and then is excessively bound with ADA in a competition reaction, so that the ADA detection method is sensitive to the target clearance, and the detection result of ADA can be obviously influenced by insufficient clearance or subtle difference, and therefore, the effect of the direct conversion of the invention patent is applied to target interference removal of ADA detection and cannot meet expectations.

In chinese patent CN115856322a, a reagent for detecting anti-o Ma Zhushan anti-drug antibodies and applications thereof are disclosed, which is characterized in that a reagent for detecting anti-o Ma Zhushan anti-drug antibodies in a sample is provided, including a pretreatment reagent, a dissociation reagent and a detection reagent, which can effectively reduce the influence of high concentration IgE, o Ma Zhushan anti-drug and IgE-o Ma Zhushan anti-complex on the determination of anti-o Ma Zhushan anti-drug antibodies. The data according to its application examples were not tested for IgE interference at concentrations above 10. Mu.g/mL. Since the extent of IgE interference with the detection method increases with increasing concentration, it cannot be speculated from the example data whether the above method is tolerant of higher concentrations of IgE interference. In allergic patients, the IgE concentration is usually between 5 mug/mL and 20 mug/mL, the method obviously cannot meet the detection requirement of all samples, and a detection method with at least 100 mug/mL of target interference resistant concentration needs to be established. Meanwhile, on ADA detection, only 0.1 mug/mL and 1 mug/mL of anti-ao Ma Zhushan anti-antibody detection is performed, and the sensitivity of the method is far from the requirement of 1ng/mL according to the calculation of P/N (positive sample measurement value/negative sample measurement value) value in the example data. Therefore, the method has more defects in the practical application process, and the conventional technical means are used for improving and optimizing the method, so that the expected requirement is difficult to reach.

In chinese patent CN115667935a, a method for using an anti-target antibody or target receptor and its cofactor to deplete a target (i.e., a target) is disclosed, thereby alleviating target interference in the detection of the anti-drug antibody, and simultaneously, test effects under a plurality of conditions, such as different pH, different anti-target antibody, different target receptor and its cofactor concentration, etc. are also explored. However, according to the data of the examples, the target tolerance concentration is about 5 mug/mL, and the requirement of detecting the anti-drug antibody of the IgE targeted drug cannot be met. Secondly, according to the application example, the anti-target antibody can even aggravate false positives in the detection process of the anti-drug antibody, and a large amount of anti-target antibody screening is needed. Meanwhile, the method for depleting the target by the receptor and the cofactor thereof is not suitable for each target, and the recombinant expression of the receptor and the cofactor thereof consistent with humanization is still a great difficulty. Therefore, the method has the advantages of high limitation, difficult popularization and large-scale application.

Meanwhile, because IgE and ADA are immunoglobulins, the two are similar in structure and physicochemical properties, and conventional physicochemical treatment modes, such as acidification, heating, PEG precipitation and the like, can possibly lead to synchronous removal of ADA and IgE.

Therefore, in view of the special situation of target interference of the IgE targeting drug and the defects of the above patents or methods, it is very necessary to develop a method for detecting the anti-drug antibody of the IgE targeting drug to overcome the serious problem of target interference in the prior art and improve the sensitivity, accuracy and stability of the method for detecting the anti-drug antibody of the IgE targeting drug.

Disclosure of Invention

The invention aims to provide a detection system and a detection method for an anti-drug antibody of an IgE targeted drug, which solve the technical problems of serious target interference, low sensitivity, low accuracy and weak stability of the detection method in the prior art and realize accurate and efficient detection of the anti-drug antibody of the IgE targeted drug.

In order to achieve the above purpose, the invention adopts the following technical scheme: an IgE targeted drug antibody detection system is mainly divided into two parts: the first part is a sample processing system and the second part is a detection system.

The sample processing system is mainly used for eliminating medicine interference, target spot interference and possible interference in the ADA detection process, such as rheumatoid factor interference, matrix effect interference and the like, wherein IgE target spot interference has very critical influence on an ADA detection result, and the elimination of IgE target spot interference is an important point of the invention.

The sample processing system comprises a composite microbead reagent and a dissociating neutralizing reagent, wherein the composite microbead reagent comprises 3 mu m magnetic microbeads coupled with anti-human IgE antibodies, 30 mu m magnetic microbeads coupled with the anti-human IgE antibodies and 1 mu m polystyrene microbeads coupled with the anti-human IgE antibodies; the dissociation and neutralization reagent is common 300mM acetic acid (pH2.00.+ -. 0.05) and 1M Tris solution (pH9.5.+ -. 0.5).

The sample processing system comprises a pretreatment process and a post-treatment process, wherein the pretreatment process is to use magnetic microbeads in a composite microbead reagent to grasp and separate IgE, and the pretreatment process can be called as a magnetic bead grasping process; the post-treatment process is divided into two parts, one part is to use 300mM acetic acid (pH 2.00.+ -. 0.05) to dissociate the drug-resistant antibody from the drug, and the other part is to use polystyrene microspheres in a composite microbead reagent to bind the residual IgE in the depleted sample.

The detection system comprises an ELISA plate coated with the omalizumab, biotin-labeled omalizumab, ruthenium-labeled omalizumab and an MSD plate coated with streptavidin.

The principle of the invention is as follows: the 30 mu m magnetic microbeads coupled with the anti-human IgE antibody are specifically combined with IgE in the sample, the magnetic microbeads are separated by a magnetic separation device, and more than 95% of IgE in the sample can be removed by repeating the steps, so that a sample 1 is obtained; then fully vibrating and incubating the 3 mu m magnetic microbeads coupled with the anti-human IgE antibody with the sample 1, separating the magnetic microbeads by a magnetic separation device, and further removing IgE to obtain a sample 2, wherein the IgE concentration is less than or equal to 0.5 mu g/mL; then acidifying the sample 2 by using 300mM acetic acid to dissociate the omalizumab in the sample from the drug-resistant antibody to obtain a sample 3; adding the sample 3 into an ELISA plate coated with the oxmarizumab and pre-added with a 1M Tris solution, wherein the anti-drug antibody in the sample 3 is combined with the oxmarizumab coated on the ELISA plate; after washing the plate, adding 300mM acetic acid into the ELISA plate to dissociate the drug-resistant antibody and the oxmarizumab coated on the ELISA plate, and sucking the supernatant to obtain a sample 4; mixing the sample 4 with 1M Tris solution and biotin-marked omalizumab, adding the mixture into an MSD plate (namely SA-MSD plate) coated with streptavidin, combining the drug-resistant antibody and the biotin-marked omalizumab into a compound, and connecting the compound to the MSD plate through the specific combination of biotin and streptavidin; after washing the plate, adding ruthenium marked omalizumab to form a ruthenium marked omalizumab-anti-drug antibody-biotin marked omalizumab complex; after multiple plate washes, data reading and analysis were performed using an MSD plate reader. Because the concentration of the anti-drug antibody is in direct proportion to the signal value read by the instrument, the corresponding concentration result can be obtained by back calculation according to the standard test result. In addition, the detection signal value of a certain standard may be a Cutoff signal value, and then the ratio of the measurement signal value of the sample to the Cutoff signal value may be defined as COI. And outputting the final test result of the sample in a COI form, and displaying the qualitative or semi-quantitative result when the COI is less than 1 and the sample result is negative and otherwise positive.

Because the concentration of IgE in allergic patients is between 5 and 20 mug/mL, the interference concentration of target spots for detecting the anti-drug antibody of the IgE targeted drug should be at least 50 mug/mL, and in order to prevent extreme cases, the interference concentration should be preferably 100 mug/mL. Meanwhile, the generation of the anti-drug antibody can seriously interfere with the drug effect, so that the user generates immune response and even possibly endangers the life of the user, so that the higher the sensitivity of the detection method is, the more perfect the drug evaluation is, the more unknown risks possibly existing in the drug can be avoided, and the sensitivity of the method is at least 1ng/mL so as to avoid more risks.

The prior known patents and methods can not meet the requirements, and have more defects in the practical application process. In view of this problem, the inventors have made adjustments to the known patents and methods to better accommodate detection of anti-drug antibodies to IgE targeted drugs, but with the exception of a simplification in the detection steps, there is no significant improvement in the detection results, with the exception of a slight improvement, far from the expected requirements.

The separation of target IgE in a sample from a substance to be detected, namely an anti-drug antibody, is one of the best methods for solving the target interference. In the actual test process, the inventor finds that the magnetic bead grabbing is one of the better methods for removing IgE in the sample, the conventional clearance is between 40% and 60%, and the single grabbing clearance of the optimized magnetic beads can reach 70%. In the known method, only one grabbing is generally used, and the inventor provides an experimental scheme for repeatedly grabbing samples for a plurality of times in order to improve the grabbing clearance. After 9 repetitions of bead capture, the IgE concentration was reduced to undetectable and 100. Mu.g/mL of target interference was tolerated, but the sensitivity of the anti-drug antibody was at this point around 55 ng/mL. The grabbing times are reduced, the sensitivity is not obviously improved, and the capability of tolerating the interference of the target spots is obviously reduced. On one hand, the magnetic beads have a certain volume of liquid, the repeated grabbing is equivalent to diluting the sample, and meanwhile, the liquid is different from the matrix environment of the sample, so that the activity loss of the anti-drug antibodies in the sample can be caused, and finally, the overall sensitivity is reduced; on the other hand, the long time of multiple magnetic bead grabbing operation is possible, and incubation treatment is generally required to be carried out at room temperature or even 37 ℃ for about 10.5 hours, so that the activity of the anti-drug antibody is lost, and the sensitivity is further affected. Meanwhile, complicated operation steps have high operation requirements on detection personnel, and are not suitable for practical application.

The magnetic bead grabbing means that the magnetic beads coupled with the anti-IgE antibodies are mixed with a sample, incubated for 10-60 minutes, igE is grabbed on the surfaces of the magnetic beads in a mode of specific combination of the anti-IgE antibodies and the IgE, and finally the magnetic beads are separated from the sample by a magnetic separation device, so that the IgE in the sample is removed. Wherein the specific incubation time of the magnetic beads with the sample varies significantly depending on the size of the particle size of the magnetic beads and the time required to achieve optimal clearance.

To solve the above problems, the inventors selected magnetic beads of different particle diameters for more efficient separation of IgE from a sample, and performed a grab separation of IgE in the sample, including 3 μm magnetic beads coupled with anti-human IgE antibodies and 30 μm magnetic beads coupled with anti-human IgE antibodies.

The 30 μm magnetic beads are the largest diameter and more common magnetic beads known in the market, and the process is mature and stable. Although the specific surface area of the large-diameter magnetic beads is small, the amount of adsorbable IgE is far smaller than that of the small-diameter magnetic beads, the single grabbing clearance rate is low, but the contact surface of the large-diameter magnetic beads with IgE is large due to the large surface area of the single magnetic beads, the self optimal adsorption amount can be achieved in a short time, and meanwhile, the clearance efficiency is considerable due to the higher concentration of IgE in a sample. The small-diameter magnetic beads have small surface area, and the magnetic beads are fully dispersed and mixed in a sample to ensure that the magnetic beads are fully combined with IgE, so that the optimal clearance rate is achieved, and the single grabbing time is long. Considering the practical situation, the magnetic beads with the particle size of 30 mu m are selected to be used for grabbing the sample for 3 times, each time is incubated for 10 minutes, grabbing of the magnetic beads with the large particle size is completed within about 40 minutes, and through experimental measurement, more than 90% of IgE can be removed in the step. The number of grabs was then increased continuously and the expected effect was not achieved, even when the IgE concentration reached 1. Mu.g/mL, without significant change. This is probably because, on the one hand, the removal efficiency of the magnetic beads with large particle diameters is not high originally, but has a relatively obvious removal effect under the condition of high concentration of IgE, and when the IgE concentration is reduced, the removal efficiency returns to normal; on the other hand, the large-diameter magnetic beads have too large volume per se, cannot be fully dispersed in a sample to be combined with IgE, and the clearance efficiency is extremely low after the IgE is lower than a certain concentration.

To better remove the remaining IgE, the inventors selected a 3 μm magnetic bead to further grasp the IgE. Compared with the magnetic beads with the thickness of 30 mu m, the specific surface area of the magnetic beads with the thickness of 3 mu m is improved by 10 times, and the magnetic beads with the thickness of 3 mu m are easier to disperse in a sample; the single beads are more grippable and easier to separate and require shorter incubation times to achieve optimal clearance than the 1 μm and below beads. In practical tests, samples with IgE concentration of 5. Mu.g/mL were grasped 2 times using 3 μm magnetic beads, and incubated for 60 minutes each time, at which the IgE concentration was approximately between 200ng/mL and 500 ng/mL.

When the concentration of IgE is below 500ng/mL, the IgE can be further eliminated by continuing the grabbing separation by using the magnetic beads with the diameter of 3 mu m or less, but the efficiency is extremely low, the time consumption is long, and the optimization of the whole detection result is not remarkable. It is possible that part of IgE in the sample is combined with proteins such as drugs or receptors to form a combined state, and cannot be efficiently captured by the magnetic beads. The introduction of a dissociating agent during the bead gripping phase may possibly increase the removal efficiency, but repeated acidolysis and neutralization may further increase the risk of loss of activity of the drug-resistant antibody. Thus, the inventors believe that during the bead grasping phase, extending sample processing time and introducing a dissociating agent to thoroughly remove IgE in the sample may be detrimental to detection of the anti-drug antibodies. Therefore, the addition of high concentrations of anti-IgE antibodies or IgE receptors to the sample to bind IgE to the sample during incubation thereof is considered to deplete IgE as much as possible, possibly reducing or even eliminating the impact of detection of the anti-drug antibodies. When the concentration of the anti-IgE antibody or IgE receptor reaches 1ug/mL, the interference of IgE is obviously reduced, but the concentration is continuously increased, the detection result is not obviously affected, and in the test process, false negative reaction and false positive reaction can be generated. The false negative results are due to: the immune complex formed by the combination of IgE receptor and IgE can still be combined with a capture reagent or a detection reagent in the detection process to form competition with the anti-drug antibody. The false positive results are due to: the anti-IgE antibody forms a complex immune complex after binding to 2 IgE, similar to an anti-drug antibody, and can be linked to both a capture reagent and a detection reagent to generate a positive signal.

To solve this problem, the inventors selected 1 μm polystyrene microspheres to which anti-human IgE antibodies were added, and during the reaction, the microspheres were first bound to IgE in the sample, and the ability of IgE bound to the microspheres to compete with the anti-drug antibodies for binding to omalizumab was significantly reduced due to steric hindrance of the microspheres, and the influence on detection of the anti-drug antibodies was significantly reduced. And compared with magnetic beads, the polystyrene microsphere does not contain ferric oxide, has better suspension property in a liquid phase, is not easy to settle in the reaction process, and can provide whole-course protection for the reaction. Insufficient steric hindrance can exacerbate false positive or false negative results if the microbead diameter is too small. When the diameter is too large, dispersibility tends to be low, and as a result, stability tends to be low.

Therefore, a preferred IgE interference elimination protocol of the present invention is to select composite microbeads to resist IgE interference during detection, wherein 30 μm magnetic microbeads coupled with anti-human IgE antibodies and 3 μm magnetic microbeads coupled with anti-human IgE antibodies are used to grasp IgE in the isolated sample during sample pretreatment to reduce its concentration to below 0.5 μg/mL within 3 hours. And then during the post-treatment of the sample, the 1 mu m polystyrene microsphere coupled with the anti-human IgE antibody is used for further combining and consuming the free IgE in the sample, so that the IgE is prevented from interfering with the detection result. Finally, the method can resist the interference of 100 mug/mL IgE in the sample, and the detection sensitivity can reach 0.69ng/mL.

The inventor adopts two ways to solve the problem because the liquid carried by the magnetic beads can dilute the anti-drug antibodies in the sample and change the matrix environment of the sample, thereby causing the detection sensitivity of the anti-drug antibodies to be reduced. One is to use a magnetic separation device to separate the magnetic beads from the liquid before the magnetic beads are uniformly mixed with the sample, and then use the sample to re-suspend the magnetic beads, so that the influence of introducing the liquid is greatly reduced. On the other hand, the magnetic beads are freeze-dried on the surface of a 96-well plate, and the sample is used for re-dissolving the magnetic beads, so that the effect can be achieved.

The invention also provides a detection method of the anti-drug antibody of the IgE targeted drug, which eliminates IgE in the sample.

Specifically, the IgE in the sample is eliminated by using a magnetic bead.

Preferably, the IgE in the sample is eliminated using composite microbeads, including 3 μm magnetic microbeads of anti-human IgE antibodies, 30 μm magnetic microbeads of anti-human IgE antibodies, and 1 μm polystyrene microbeads of anti-human IgE antibodies.

Preferably, the method for detecting the anti-drug antibody comprises a pretreatment process and a post-treatment process, wherein the pretreatment process is to use magnetic microbeads in a composite microbead reagent to grasp and separate IgE, and the pretreatment process can be called as a magnetic bead grasping process; the post-treatment process is divided into two parts, one part is to use 300mM acetic acid (pH 2.00.+ -. 0.05) to dissociate the drug-resistant antibody from the drug, and the other part is to use polystyrene microspheres in a composite microbead reagent to bind the residual IgE in the depleted sample.

Compared with the prior art, the invention has the following beneficial effects: the system and the method are used for detecting the anti-drug antibody of the IgE targeted drug, and the detection sensitivity can reach 0.69ng/mL.

Detailed Description

The technical scheme of the invention will be fully described in the following by combining examples.

Example 1: magnetic bead coupled anti-IgE antibodies of different diameters

1.1 taking 1mL of 30 μm carboxyl magnetic beads and 1mL of 3 μm carboxyl magnetic beads respectively, adding the beads with a solid content of 10mg into a centrifuge tube, standing on a magnetic separator for 2min, and discarding the supernatant, wherein specific reagent information is shown in the following table 1.

TABLE 1

Reagent name	Suppliers (suppliers)	Goods number
			30 mu m carboxyl magnetic bead	Dongna organism	MB1023
3 mu m carboxyl magnetic bead	Dongna organism	MB1029

1.2 adding 1mL of activation buffer (50 mM MES, pH 6.0), shaking and mixing, standing on a magnetic separator for 2min, discarding supernatant, and repeating washing for 3 times;

1.3 adding 900. Mu.L of an activation buffer (50 mM MES, pH 6.0) to resuspend the microspheres, mixing by vortexing;

1.4 adding 50 mu LEDC (40 mg/ml) and 50 mu L NHS (40 mg/ml) solution, mixing by vortex shaking, and incubating at 37deg.C for 30min in dark place by shaking;

1.5, after incubation, standing on a magnetic separator for 2min, and discarding the supernatant;

1.6 adding 1mL of activation buffer (50 mM MES, pH 6.0), mixing by vortex shaking, standing on a magnetic separator for 2min, discarding the supernatant, and repeating washing for 3 times;

1.7 adding 500. Mu.l of an activation buffer (50 mM MES, pH 6.0), mixing by vortexing, adding 100. Mu.g of an anti-IgE antibody per mL of microspheres, and finally adding the activation buffer until the total volume is 1mL, and incubating for 2h at 37 ℃ by shaking in a dark place;

1.8, after incubation, standing on a magnetic separator for 2min, and discarding the supernatant;

1.9 adding 1mL of blocking solution (containing 5% BSA and 4% glycine), stirring, mixing, and incubating at 37deg.C in dark place for 1 hr;

1.10, after incubation, standing on a magnetic separator for 2min, and discarding the supernatant;

1.11 adding 1mL of washing buffer (containing 0.1% Tween-20), mixing by vortex oscillation, standing on a magnetic separator for 2min, discarding supernatant, and washing repeatedly for 2 times;

1.12 mL of protein stabilizing solution (containing 2% BSA and 2% trehalose) is added, vortex shaking and mixing are carried out, and the mixture is preserved at 2-8 ℃ in a dark place, thus obtaining 2 magnetic microspheres coupled with anti-IgE antibodies with different diameters.

Example 2: polystyrene microsphere coupled anti-IgE antibody

2.1 taking 1mL of polystyrene microsphere with a solid content of 10mg, adding into a centrifuge tube, centrifuging at 12000rpm for 15min by a high-speed centrifuge, and discarding the supernatant, wherein the specific reagent information is shown in Table 2.

TABLE 2

Reagent name	Suppliers (suppliers)	Goods number
			950nm polystyrene microsphere (carboxyl)	Dongna organism	XHPS950

2.2 adding 1mL of activation buffer (50 mM MES, pH 6.0), shaking by vortex, mixing, centrifuging at 12000rpm for 15min by high-speed centrifuge, discarding supernatant, and repeating washing for 3 times;

2.3 adding 900. Mu.L of activation buffer (50 mM MES, pH 6.0) to resuspend the microspheres, mixing by vortexing;

2.4 adding 50 mu LEDC (40 mg/ml) and 50 mu L NHS (40 mg/ml) solution, mixing by vortex shaking, and incubating at 37deg.C for 30min in dark place by shaking;

2.5 after incubation, centrifuging at 12000rpm for 15min by a high-speed centrifuge, and discarding the supernatant;

2.6 adding 1mL of activation buffer (50 mM MES, pH 6.0), mixing by vortex shaking, centrifuging at 12000rpm for 15min by high-speed centrifuge, and discarding the supernatant;

2.7 adding 500. Mu.l of activation buffer (50 mM MES, pH 6.0), mixing by vortexing, adding 100. Mu.g of anti-IgE antibody per mL of microsphere, adding activation buffer to 1mL of total volume, and incubating for 2h at 37deg.C in dark shaking;

2.8, after incubation, centrifuging at 12000rpm for 15min by a high-speed centrifuge, and discarding the supernatant;

2.9 adding 1mL of blocking solution (containing 5% BSA and 4% glycine), stirring, mixing, and incubating at 37deg.C in dark place for 1 hr;

2.10 after incubation, centrifuging at 12000rpm for 15min by a high-speed centrifuge, and discarding the supernatant;

2.11 adding 1mL of washing buffer (containing 0.1% Tween-20), mixing by vortex shaking, centrifuging at 12000rpm for 15min by a high-speed centrifuge, discarding the supernatant, and washing repeatedly for 2 times;

2.12 adding 1mL of protein stabilizing solution (containing 2% BSA and 2% trehalose), stirring, shaking, mixing uniformly, and preserving at 2-8deg.C in dark to obtain polystyrene microsphere coupled with anti-IgE antibody.

Example 3: marking biotin or ruthenium by omalizumab

3.1 pre-dissolving water-soluble biotin or ruthenium to a final concentration of 10mg/ml;

3.2, respectively adding the omalizumab into a desalting column or an ultrafiltration tube, and centrifugally collecting the omalizumab in a centrifuge tube;

3.3, adding biotin or ruthenium solution according to the mol ratio of the omalizumab to the biotin or ruthenium of 1:20, and uniformly mixing with the omalizumab;

3.4 Incubating overnight at 4deg.C in the absence of light;

3.5, adding the same volume of Tris-HCl after the incubation is finished, and incubating for 1h at room temperature in a dark place;

3.6 after incubation, removing unreacted biotin by a desalting column or an ultrafiltration centrifuge tube;

3.7 measuring the concentration of the desalted antibody by using an ultraviolet spectrophotometer to obtain biotin-labeled or ruthenium-labeled omalizumab, and storing at-20 ℃.

Example 4: other related solution configurations

4.1 preparation of phosphate buffer: to 1000mL of pure water, 3.392 + -0.034 g of disodium hydrogen phosphate dodecahydrate, 0.083+ -0.001 g of sodium dihydrogen phosphate dihydrate and 8.766 + -0.088 g of sodium chloride were added in this order, and the mixture was thoroughly dissolved and mixed, sterilized and filtered, and then stored at room temperature.

4.2 preparation of experiment buffer: to 1000mL of phosphate buffer, 20.0000.+ -. 0.2g of BSA and 0.3mL ProClinTM300 were added in this order, and the mixture was thoroughly dissolved. Sterilizing, filtering, and storing at 2-8 deg.c.

4.3 preparation of washing buffer: to 1000mL of pure water, 33.92 + -0.34 g of disodium hydrogen phosphate dodecahydrate, 0.827 + -0.008 g of sodium dihydrogen phosphate dihydrate and 8.766 + -0.088 g of sodium chloride were added in this order, and then 0.5mL of triamcinolone acetonide-100 was added thereto, and the mixture was sufficiently dissolved and mixed. Sterilizing, filtering, and storing at room temperature.

4.4 preparation of a sealing liquid: to 1000mL of phosphate buffer, 30.0000.+ -. 0.3g of BSA and 0.3mL of LProClinTM300 were added in this order, and the mixture was thoroughly dissolved and mixed. Sterilizing, filtering, and storing at 2-8 deg.c.

4.5 Preparation of 1M Tris solution: to 300mL of pure water, 36.34.+ -. 0.36g Trizma@base was added, and the mixture was dissolved and mixed thoroughly, sterilized and filtered, and then stored at room temperature.

4.6 Preparation of 300mM acetic acid solution: to 580mL of pure water, 10mL of glacial acetic acid was added, and the mixture was thoroughly dissolved and mixed, sterilized and filtered, and then stored at room temperature.

4.7 preparation of microsphere working solution: to 990. Mu.L of the test buffer, 10. Mu.L of the polystyrene microspheres of example 2 were added, mixed thoroughly, stored at 2-8℃and mixed with shaking again before use.

4.8 preparation of Read Buffer: 200mL of 4 XRead Buffer was added to 200mL of pure water, and the mixture was dissolved and mixed thoroughly, and stored at room temperature.

Example 5: igE capture clearance test of magnetic beads

5.1 adding high concentration IgE into healthy human serum to make the final concentration be 100 mug/mL, wherein the serum content is not less than 95%, detecting the sample by using an immunoglobulin E (IgE) detection kit (immune transmission turbidimetry) produced by Shenzhen Mierei biomedical electronics Co., ltd.), diluting the sample according to the estimated concentration according to the instruction before the detection by corresponding times, and obtaining the concentration of 100.51 mug/mL according to 1 IU=2.42 ng after the IgE concentration in the serum is detected to 41531.31 IU/mL.

5.2 magnetic beads of different diameters, including 30 μm, 20 μm, 10 μm, 5 μm, 3 μm and 1 μm, were coupled as in example 1.

5.3 determination of optimal incubation time for magnetic beads of different particle sizes: the magnetic beads of example 1 were diluted 10-fold with phosphate buffer to give a magnetic bead dilution, then 10. Mu.L of the magnetic bead dilution was taken in a 96-well plate, the magnetic beads were separated from the liquid with a magnetic separator, and 100. Mu.L of the sample was added thereto, mixed with shaking, and incubated. After incubation for different times (i.e. 0min, 10min, 20min, 30min, 45min, 60min, 90min, 120 min), the magnetic beads were separated, and the supernatant was aspirated and detected using an immunoglobulin E (IgE) assay kit (immunonephelometry) manufactured by Shenzhen Mei biomedical electronics Co., ltd.) to obtain the concentration of IgE. The control group was blank magnetic beads of different diameters to which no anti-IgE antibody was added during the coupling process. The results show that the optimal incubation time of the magnetic beads with different particle sizes is obviously different, the magnetic beads can reach saturation in 10 minutes at the shortest and reach saturation in 60 minutes at the longest, and the blank magnetic beads have almost no adsorption to IgE. The detailed data analysis results were as follows:

5.4 verification of the effect of multiple grabbing of magnetic beads with the particle size of 3 μm: according to the result of 5.3, the IgE removal effect of the magnetic beads with the particle size of 3 μm after incubation with the sample for 60min is best, repeated grabbing test is carried out according to the operation step of 5.3, the mixed incubation time is 60min each time, the steps are repeated for at most 9 times, and after separation for the 1 st, 3 rd, 5 th, 7 th and 9 th times, the supernatant is sucked and detected by using an immunoglobulin E (IgE) measuring kit (immunotransmittance turbidimetry) produced by Shenzhen biological medical electronics Co., ltd, so that the concentration of IgE is obtained. The control group was a 3 μm particle size blank magnetic bead without anti-IgE antibody added during the coupling process. The results showed that after 9 grabs, igE was not detected and the overall treatment time was 10.5h. The detailed data analysis results were as follows:

5.5 verification of the magnetic bead grabbing effect: according to the procedure of 5.3, samples were grasped 3 times using 30 μm particle size magnetic beads and mixed for 10min each incubation. After the completion, the sample is grabbed for 2 times by magnetic beads with the particle size of 3 mu m, the mixing incubation time is 60min each time, finally, the supernatant is sucked and detected by using an immunoglobulin E (IgE) detection kit (immune transmission turbidimetry) manufactured by Shenzhen biomedical electronics Co., ltd, the concentration of IgE is 0.32 mu g/mL, and the whole treatment time is 3h.

Example 6: establishment of IgE targeted drug antibody detection method

6.1 coating: the omalizumab was diluted to 5 μg/mL using phosphate buffer, 100 μl was added per well in a 96 well assay plate, and incubated with sealing plate membrane sealing plates with shaking at 300rpm for 120±10 minutes at room temperature.

6.2 closing: the 96-well assay plate was removed, washed 3 times with 300. Mu.L/well wash buffer, and dried on absorbent paper. 200. Mu.L/well of blocking solution was added for blocking, membrane-sealing plates were used for sealing, and incubation was performed for 1-3 hours at room temperature with shaking at 300 rpm.

6.3 sample pretreatment: the sample to be tested was subjected to pretreatment in the manner of example 5 to obtain a treated sample. The sample to be measured comprises: positive quality control, negative quality control, sensitivity curve samples, unknown samples, and the like.

6.4 first acidification treatment: a dilution plate was taken, 50. Mu.L/well of the treated sample was added to the plate according to the plate layout, and then 50. Mu.L/well of 300mM acetic acid was added thereto, and the plate was sealed with a sealing plate membrane, and the plate was dissociated for 10-15 minutes at room temperature at 300 rpm.

6.5ADA grasp incubation: the blocked 96-well assay plate was removed, the well liquid was discarded, and the plate was washed 3 times with 300. Mu.L/well wash buffer and then dried. To a 96-well assay plate, 10. Mu.L/well of 1M Tris solution was added, then 80. Mu.L/well of the acidified sample was added to the corresponding well, and 10. Mu.L/well of polystyrene microsphere working solution was added, and the plate was sealed with a sealing plate and incubated overnight at 800rpm at room temperature for 16-20 hours.

6.6 second acidification treatment: the neutralized 96-well assay plate was removed, the well liquid was discarded, and the plate was washed 3 times with 300. Mu.L/well wash buffer and then dried by pipetting. To the corresponding wells, 80. Mu.L/well 300mM acetic acid (pH 2.00.+ -. 0.05) was added, membrane-sealed with a sealing plate and dissociated by shaking at 300rpm at room temperature for 10-15 minutes.

6.7 neutralization: the biotin-labeled omalizumab was diluted 500-fold with 1M Tris buffer to obtain a neutralization solution, then a new dilution plate was taken, 60. Mu.L of the neutralization solution was added to each well, and 60. Mu.L/well of the acidified sample was transferred to the corresponding well of the dilution plate, and neutralized and mixed well.

6.8SA-MSD plate coating: the neutralized samples were transferred to SA-MSD assay plates at 100. Mu.L/well. The plates were sealed and incubated with shaking at 300rpm for 1 hr.+ -. 10min.

6.9 incubation of detection antibody: and (3) carrying out 500-time dilution on the ruthenium-marked omalizumab by using an experiment buffer solution to obtain a detection working solution. The SA-MSD assay plates were washed 3 times with 300. Mu.L/well wash buffer and blotted dry on absorbent paper. 100 mu L of detection working solution is added into each hole, membrane sealing plates are used for sealing plates, and the incubation is carried out for 60-90 minutes at room temperature and 300 rpm.

6.10 reading the plate: the incubated SA-MSD assay plate was removed, the liquid in the wells was discarded, the plate was washed 3 times with 300. Mu.L/well wash buffer and then dried by pipetting. Read Buffer was added to the plate at 150. Mu.L/well for reading. The reading was completed within 10 minutes.

6.11 description: the detection method consists of a screening experiment and a confirmation experiment, wherein the steps are screening experiment steps. The confirmation step is different from the screening experiment step in that 500 mug/mL of omalizumab needs to be added into the detection working solution in the 6.8 detection incubation process. Confirmation experiments were used to circumvent false positive reactions that may occur in screening experiments.

Example 7: establishment of sensitivity of anti-drug antibody detection method of IgE targeted drug

7.1: in the experiment, mixed human serum is used for preparing sensitivity curve samples, the concentration of the anti-drug antibodies is respectively 500.00ng/mL, 166.67ng/mL, 55.56ng/mL, 18.52ng/mL, 6.17ng/mL, 2.06ng/mL and 0.69ng/mL, and the concentration of target IgE is 100 mug/mL, wherein the humanized anti-drug antibodies are difficult to obtain and are replaced by rabbit anti-omazumab polyclonal antibodies. After samples were processed using different sample pretreatment methods, they were screened and checked using the method of example 6 to confirm the detection sensitivity.

7.2: the experimental groups are divided into 6 groups, and the pretreatment mode of A, B, C groups of samples is the same as that of the 5.4 part in the example 5; D. e, F groups of samples were pretreated in the same manner as in section 5.5 of example 5; the control group was not subjected to sample pretreatment and was divided into G, H and I groups, which were 3 groups in total. Wherein B, E and H groups were tested using the method of example 6, in the 6.5ADA grasping step, no microsphere working fluid was added, but instead experimental buffer; C. groups F and I were tested using the method of example 6, in the 6.5ADA grasping step, no microsphere working solution was added, but instead, an experimental buffer containing 1. Mu.g/mL of anti-IgE antibody was used;

7.3: analysis of results: the sensitivity of the group D is optimal and can reach 0.69ng/mL, and obvious false positive phenomena exist in both the group C, F and the group I. The specific detection results are shown in the following table:

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example 8: method screening test precision test

8.1 experimental protocol: two test persons performed 6 experiments of analysis batches within 3 days, each batch comprising 6 independent pretreatment negative quality controls (NC), low concentration positive quality controls (LPC), medium concentration positive quality controls (MPC) and high concentration positive quality controls (HPC). Sample pretreatment was performed as in section 5.5 of example 5 and detection was performed as in example 6.

8.2 evaluation criteria: because of certain difference of signal values tested each time, the precision in the batch is determined by the S/N of HPC, MPC, LPC in the analysis batch, the precision among the batches is determined by the S/N of all analysis batches, the signal value size relationship needs to meet that HPC > MPC > LPC is larger than or equal to NC, the signal value CV in the batch of NC needs to meet less than or equal to 10%, and the signal value CV among the batches needs to meet less than or equal to 15%.

8.3 experimental results: the method has the screening experiment precision less than 10%, and detailed experiment results are shown in the following table:

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example 9: lyophilization of magnetic beads

9.1 preparation of lyophilization buffer: to 1000mL of phosphate buffer, 20.+ -. 0.2g of BSA, 5.+ -. 0.05g of trehalose, 2 g.+ -. 0.02g of mannitol, 0.1mL of Tween-80 and 0.3mL ProClinTM300 were added in this order, and the mixture was thoroughly dissolved and mixed. Sterilizing, filtering, and storing at 2-8 deg.c.

9.2 preparation before lyophilization: the magnetic beads were 50-fold diluted with a lyophilization buffer to obtain microsphere lyophilized solution, and then 50. Mu.L/well microsphere lyophilized solution was added to a 96-well plate and placed in a lyophilizer.

9.3 lyophilization procedure set up: the specific lyophilization procedure is set forth in the following table:

segment number	Target temperature (. Degree. C.)	Heating time (min)	Target vacuum	Constant temperature time (h)
					1	-50	0	0	1.5
2	-15	0	0	1
					3	-50	0	0	3
4	-45	30	0	2
					5	-40	45	0	4
6	-35	60	0	6
					7	-30	60	0	8
8	-25	45	0	4
					9	-15	30	0	1
10	-5	0	0	1
					11	4	0	0	1
12	15	0	0	1
					13	25	0	0	8
14	4	0	0	24

The vacuum pump is started in section 3, the freeze dryer lyophilization chamber is evacuated and maintained in vacuum throughout the lyophilization stage. After the procedure has entered section 14, capping may be performed. And after the completion, restoring normal pressure, opening a vacuum pump, taking out the 96-well plate, placing the 96-well plate and the drying agent into an aluminum foil bag for plastic package, and placing the aluminum foil bag into a temperature of 2-8 ℃ for preservation.

The invention and its embodiments have been described above without limitation, and the actual construction is not limited thereto. In summary, if one of ordinary skill in the art is informed by this disclosure, a structural manner and an embodiment similar to the technical solution should not be creatively devised without departing from the gist of the present invention.

Claims (10)

1. An IgE targeted drug anti-drug antibody detection system comprising a sample processing system and a detection system, wherein the sample processing system comprises microbeads coupled with anti-human IgE antibodies and a dissociating neutralizing agent; the detection system comprises an ELISA plate coated with the omalizumab, biotin-labeled omalizumab, ruthenium-labeled omalizumab and an MSD plate coated with streptavidin.

2. The IgE targeted drug anti-drug antibody detection system of claim 1, wherein the microbeads are composite microbeads, including magnetic microbeads and polystyrene microbeads.

3. The IgE targeted drug anti-drug antibody detection system of claim 1, wherein the microbeads are composite microbeads comprising 3 μm magnetic microbeads of anti-human IgE antibodies, 30 μm magnetic microbeads of anti-human IgE antibodies and 1 μm polystyrene microbeads of anti-human IgE antibodies.

4. The IgE targeted drug anti-drug antibody detection system of claim 1, wherein the microbeads are separated from the liquid prior to use.

5. The IgE targeted drug resistant antibody detection system of claim 1, wherein the dissociative neutralizing agent is acetic acid and Tris solution.

6. A method for detecting an anti-drug antibody of an IgE targeted drug, which is characterized in that IgE in a sample is eliminated before detection.

7. The method for detecting an IgE-targeted drug antibody according to claim 6, wherein the IgE in the sample is eliminated by using magnetic beads.

8. The method for detecting an IgE-targeted drug antibody according to claim 7, wherein IgE in the sample is eliminated by using composite microbeads including 3 μm magnetic microbeads of an anti-human IgE antibody, 30 μm magnetic microbeads of an anti-human IgE antibody and 1 μm polystyrene microbeads of an anti-human IgE antibody.

9. The method for detecting an IgE-targeted drug antibody according to claim 7, comprising a pretreatment process and a post-treatment process, wherein the pretreatment process is to use magnetic microbeads in a composite microbead reagent to grasp and separate IgE, and the pretreatment process can be called a magnetic bead grasping process; the post-treatment process is divided into two parts, one part is to use 300mM acetic acid (pH 2.00.+ -. 0.05) to dissociate the drug-resistant antibody from the drug, and the other part is to use polystyrene microspheres in a composite microbead reagent to bind the residual IgE in the depleted sample.

10. The method for detecting an IgE-targeted drug antibody according to claim 9, wherein the detection sensitivity is up to 0.69ng/mL.